Effect of introducing disulfide bridges in C-terminal structure on the thermostability of xylanase XynZF-2 from <i>Aspergillus niger</i>

Abstract

In this study, a mutant xylanase of high thermostability was obtained by site-directed 1 mutagenesis. The homologous 3D structure of xylanase was successfully modeled and the mutation 2 sites were predicted using bioinformatics software. Two amino acids of XynZF-2 were respectively 3 substituted by cysteines (T205C and A52C) and a disulfide bridge was introduced into the C-terminal 4 of XynZF-2. The mutant gene xynZFTA was cloned into pPIC9K and expressed in P. pastoris. The 5 optimum temperature of the variant XynZFTA was improved from 45 o C to 60 o C, and XynZFTA 6 retained greater than 90.0% activity (XynZF-2 retained only 50.0% activity) after treatment at 50oC for 7 5 min. The optimum pH of mutant xylanase was similar to XynZF-2 (pH=5.0). The pH stability span 8 (5.0~7.0) of the mutant xylanase was increased to 3.0~9.0. Overall, the results implied that the 9 introduction of a disulfide bridge in the C-terminal structure improved the thermostability and pH 10 stability of XynZF-2.

Simultaneously Enhanced Thermostability and Catalytic Activity of Xylanase from Streptomyces rameus L2001 by Rigidifying Flexible Regions in Loop Regions of the N-Terminus

“…However, the introduction of additional disulfide bonds in these regions might confer greater rigidity to the enzyme structure than intermolecular interactions, such as hydrogen bonds, but also carried a higher risk of causing a greater loss of flexibility in the enzyme structure and thus a reduction in catalytic activity. [30][31][32][33]48 According to the present results, the addition of hydrogen bonds and hydrophobic interactions in the N-terminal loop region of xylanase by combination mutation could enhance the rigidity of the enzyme structure and maintain a certain degree of flexibility (Figure 5), thus achieving a coupled increase in enzyme thermal stability and catalytic activity (Table 1 and Figure 2). The present results suggested that the N-terminal flexible loop region was not only an effective target for enhancing GH11 xylanase but could also be a potential site for the improvement of its catalytic activity.…”

“…Focused analyses of the mutated regions of the xylanase mutants revealed enhancements in hydrogen bonds and hydrophobic interactions. Including a new hydrophobic interaction formed between Thr 11 and Phe 16 , a new hydrophobic interaction formed between His 12 and Leu 31 , a new hydrogen bond formed between His 12 and Gly 32 , two new hydrogen bonds formed between Asp 13 and Pro 33 and Gly 34 , two new hydrogen bonds formed between Tyr 15 and Ser 190 and Ser 191 , and a new hydrogen bond formed between Gly 32 and Lys 51 , respectively (Figure 3). In addition, the introduction of 11 YHDGYF 16 , 23 AP 24 / 23 SP 24 , and 32 GP 33 to Mut1 and Mut2 changed the surface electrostatic potential from neutral to negative (Figure 4).…”

Cloning, Expression, and Characterization of a GHF 11 Xylanase from <i>Alteromonas macleodii </i>HY35<i> </i>in <i>Escherichia col</i><i>i</i>

“…The amino acids of the protein active center were predicted through the website (https://www.rcsb.org/). The homology and evolutionary tree of the sequence were processed with DNAMAN 9.0 (Cai et al, 2019).…”